Project Summary/Abstract The synthesis of full-length, functional proteins from completely synthetic peptides is a long-standing goal of chemistry and biology, and would allow for the incorporation of multiple non-canonical amino acids (NCAAs) to probe structure and function. Although peptide synthesis can routinely yield molecules ~50 amino acids in length, techniques like native chemical ligation (NCL) are necessary to link them into larger molecules. To date, however, NCL has been limited to proteins 150-200 residues in length, and membrane proteins are particularly challenging due to their hydrophobic nature. We propose to develop a novel method for stepwise NCL templated by DNA splints, which will enhance the local concentration of every peptide coupling step, and allow for the synthesis of significantly longer molecules than traditional NCL. Our method will be applied to make complex targets like G- protein coupled receptors (GPCRs) by using detergents or lipid bilayers to solubilize the component peptides and allow them to fold into their native conformation. The key innovation in our method is the use of DNA-peptide- DNA triblock molecules. The two orthogonal DNA handles will serve as both solubilizing tags (particularly useful for hydrophobic membrane protein peptides), and to enhance the coupling of two peptides by using a selective splinting strand to bring them into close proximity. The N-terminal handle will be attached via a photocleavable thiol auxiliary, and the C-terminal handle via a thioester. When the splint brings the N-terminus of one peptide into close proximity with the C-terminus of the next peptide, NCL will occur spontaneously due to the increased local concentration. Cleavage of the DNA tags with UV light will then leave a fully native amide bond behind. This process can be repeated for each subsequent peptide, and because the DNA will enhance the rate of coupling by co-localizing the termini, there should be no drop-off in yield of the couplings with each step. Our method will be optimized on short model peptides, followed by synthesis of small and then larger proteins, and finally GPCRs. We will introduce multiple NCAAs into the proteins to aid in experiments like FRET, EPR, or coupling to additional materials. Specific technological innovations and molecular targets include: (1) Synthesizing DNA-peptide-DNA triblock molecules linked with orthogonal, cleavable DNA handles at their termini, (2) Optimization of the method on short peptides, small proteins like lysozyme, model systems like GFP, as well as larger proteins like ovalbumin, which are beyond the capabilities of traditional NCL, (3) Application of the method to membrane proteins like GPCRs, which are highly important targets for drug development as well as fundamental biology, but are difficult to crystallize or characterize with other methods, (4) Immobilization of GPCRs on DNA origami scaffolds through the introduction of multiple, site-specific oligonucleotide handles, for use as fiducial markers for cryo-electron microscopy. The proposed work will enable the synthesis of significantly longer proteins from synthetic peptides, and has the potential to revolutionize multiple fields, as well as provide a paradigm shift for GPCR and other membrane protein structural biology.